The present invention relates to the in vitro culture of animal cells and, more particularly, to an apparatus for providing oxygen to animal cells which are in suspension culture with culture medium in a suspension culture vessel.
The in vitro culture of animal cells, particularly for purposes of recovering proteins either normally secreted by such cells or secreted by such cells by virtue of manipulation of their genetic machinery, has assumed increasingly greater prominence as a consequence of the increasing need for large quantities of proteins for therapeutic, diagnostic and investigative purposes, and the recognition that animal cells (per se. or as a hybrid partner, or as a host for an exogeneous gene) offer the best source of proteins which are the same as or closely similar o those actually employed by animals (e.g., humans) in vivo in carrying out regulatory, immune response, and other like functions.
Despite the recognized advantages of, and needs for, in vitro animal cell culture, the culture of cells outside the animal body is a difficult proposition at best, made even more difficult by the present-day demand that such processes be carried out efficiently and economically so as to achieve ultimate protein products which are not unreasonably expensive. The ultimate aim of in vitro animal cell culture processes is to provide the cells with an environment which closely mimics that which the cells are exposed to in vivo, in terms, e.g., of nutritional requirements, oxygen requirements, temperature, pH, carrying away of wastes, etc., thereby permitting the cells to grow, behave and produce product as they would in vivo, with the added burden of attempting to mimic this environment in larger scale than the microenvironment which normally would be present, for these cells, in the animal itself. At least in theory, it is possible to devise elaborate in vitro systems involving simulations of capillaries, lungs, kidneys and the like to provide the requisite environment, but often not in any remotely cost-effective manner.
A great many in vitro animal cell culture devices and systems are known in the art for culture of both anchorage-dependent cells and cells which can be grown without need for attachment to a substrate. For the culture of animal cells on a sufficiently large scale so as to be potentially suitable for mass production of proteins, among the more popular choices of culture devices is a fermenter in which cells are cultured in suspension in an appropriate culture medium. Anchorage-dependent cells also can be cultured in this way by affixing them to suitable substrate surfaces in the nature of microcarrier particles. Fermenters can be operated on a batch, semi-batch or continuous (perfusion) basis, with periodic or continuous removal of medium from the vessel and replacement with fresh culture medium.
Among the most important "nutrients" for animal cells is oxygen, and the provision of means for supplying the required degree of oxygen to the culturing cells in a suspension culture device poses great difficulties which severely restrict the ability to conduct suspension culture processes at the large-scale (i.e., to support growth of a large number of cells) required to produce ultimate protein products economically.
One means for supplying oxygen to cells in suspension culture is by means of surface aeration, i.e., providing oxygen or oxygen-containing gas in the headspace, above the culture medium level, in the closed culture system. Generally, however, the rate at which oxygen can diffusively transfer from the gas phase to the liquid phase in such systems is relatively low and, thus, growth and maintenance of only a relatively small number of cells can be supported in this manner, relegating it to utility only in small suspension culture vessels. The rate of gas transfer can be increased if the liquid phase is agitated (e.g., as in a stirred reactor), but the increase in gas transfer achievable in this manner is not so great as to offer utility in anything other than relatively small systems.
Another means for providing oxygen to cells in suspension culture is to bubble gas directly through the culture (direct sparging). Apart from providing oxygen to the cells, direct sparging can also be relied upon to bring about circulation of the suspension in the vessel, e.g., based upon fluidizing principles or draft tube devices and the like. While direct sparging is a very efficient means of oxygenation, it generally is very damaging to animal cells. Also, sparging leads to foam formation which itself can damage the cells. Although the use of surfactants can eliminate foam formation, the presence of the surfactant in the eventually harvested culture medium can lead to very difficult and expensive problems in purification of the desired secreted protein product.
It also has been proposed to provide oxygen to cells in suspension culture medium by indirect sparging, i.e., passing oxygen into or on one side of a gas-permeable (but generally liquid-impermeable) tube or membrane arranged in the medium (e.g., silicone rubber tubes or sheets), and through or across which the oxygen permeates into the culture medium. It is generally possible in this way to achieve bubble-free and foam-free aeration, but such methods generally do not provide sufficient gassing efficiency to support the growth of a large number of cells in stirred reactors, due to a combination of moderate gas transfer coefficients (less than about 0.1 cm/min in stirred reactors) and a low ratio of membrane surface to liquid volume imposed by the size and design of stirred reactors.
Another means for providing oxygen to cells in suspension culture is by sparging oxygen into the interior of a wire mesh cylinder suspended in the medium from a top cover plate over the fermentation vessel. The mesh surfaces, which are permeable to gases and liquid, tend to reduce bubbles as the gas sparged on the gas side of the mesh enters and dissolves in the liquid phase on the liquid side of the mesh, and in this way minimize damage to cells. The difficulty with such arrangements, however, is that the ability of the oxygen to efficiently transfer into the liquid phase across the screen or mesh is dependent upon the relative velocity of liquid (culture medium) in contact with the mesh on the liquid side. Thus, in order to achieve sufficient gas transfer to oxygenate the medium to the degree needed to support a large number of cells, the wire mesh cylinder must be rotated or vibrated (thereby increasing the relative velocity between the mesh surface and the liquid phase thereat). This in turn requires resort to rotating mechanical seals and/or other like moving parts which are well-known to serve as potential areas for contamination of the desired sterile environment within the fermenter.